ABSTRACT APPROACHES TO TREATING MTDNA-BASED MITOCHONDRIAL DISEASE There are no generalizable rational treatments for patients with mitochondrial diseases due to deficiency in oxidative phosphorylation (OxPhos). We propose here to study pharmacological approaches to treat patients with mitochondrial diseases due to mutations in mtDNA, based on our discovery that specific agents can shift the mutation load and/or rescue mitochondrial function in cells harboring mtDNA mutations. Specifically, we have found that the mTOR inhibitor rapamycin (sirolimus; trade name Rapamune) induces mitophagy in cells carrying homoplasmic mtDNA mutations, but does not do so in wild-type cells. Remarkably, we have now found that rapamycin treatment of heteroplasmic cells containing different pathogenic mtDNA mutations elicited a dramatic improvement in bioenergetic function, as did mdivi-1, an inhibitor of mitochondrial fission, but surprisingly, with no apparent reduction in mutant load. We now propose to follow up on these exciting results, in three ways. First, we will examine in greater detail the ability of rapamycin, mdivi-1, and other agents to restore mitochondrial function in heteroplasmic cells. Second, we will try to determine the mechanism by which functional rescue occurs, using both targeted (e,g, genetic knockdown) and unbiased (e.g. microarray) approaches. Third, we will assess the possibility of treating patients with rapamycin in a small clinical trial by first treating Dr. Hirano's mouse model of TK2 deficiency, which causes mtDNA depletion (synergy with Projects 1 and 3). If successful, the use of rapamycin or similar compounds could become the basis of the first rational treatment of mtDNA-based OxPhos diseases.